How To Monitor Cyanohab

Transcription

1 Application of molecular tools for routine water quality monitoring MWQI Technical Meeting May 27, 2015 Tim Otten, PhD, MPH Bend Genetics, LLC T:

2 Overview of presentation contents Brief background on CyanoHABs, sample collection/processing and genetic tools for their study Application of each method (case studies): Comparison of QPCR vs. microscopy for routine monitoring Metagenomics as a roadmap for lake monitoring Elucidate microbial community structure & function Development and validation of a geosmin QPCR assay Goal is to be able to use this information to more effectively manage our aquatic resources and to protect human and environmental health

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4 Sample collection Collection method varies by need Public health collect scum from most impacted site Environmental off shore, depth integrated (1 2 SD) Collect 0.5L in sterile plastic bottles (HDPE 2 is best) Store on wet ice in the dark until processing Don t want it to freeze which will lyse the cells Goal is to identify harmful/nuisance organisms before they reach problematic concentrations Adaptive management guides sampling frequency

5 Development of a tiered management approach to CyanoHAB monitoring WHO guidelines Experimentally derived Varies by state

6 Phytoplankton enumeration by microscopy Pros: Can inventory all phytoplankton, equipment/materials costs are low Cons: Hard to accurately count amorphous colonies, cannot distinguish toxic from nontoxic cells, relatively expensive and slow ~$125 $350/sample

7 DNA extraction and processing 3. DNA is now ready for downstream applications such as PCR/QPCR or sequencing 1. Concentrate sample 2. Extract DNA *Store filters at 20 o C until DNA extraction

8 Descent from common mcy + ancestor Mom Gloeotrichia Hapalosiphon Nostoc Microcystis Aphanizomenon Anabaena Planktothrix

9 Like many genes, toxicity and T&O are strain specific traits (not genus/species) Nontoxic Toxic 1 Abundance Core genome Time Toxic 2

10 Polymerase Chain Reacion (PCR) 1. Denature 2. Cool to anneal 3. Extend to primers Repeat 35 40X So, 1 DNA molecule after 40 cycles of PCR becomes 1.1 trillion copies [N = 1*2 (40) ] + Unknowns Amplification products can be visualized under UV light after gel electrophoresis

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12 16S 23S Phylogenetic Tree SF Microcystis sp. Web SF2 = web SF3 = web SF4 = hybrid SF5 = flake SF6 = flake SF7 = flake SF8 = flake SF9 = flake SF11 = web SF12 = web Flake Hybrid

13 Overview of real time quantitative PCR (QPCR) DNA extraction = $20 QPCR/sample/assay = $30 Pros: Provides earlier warning detection of nuisance organisms than direct measurements of toxins or cell counting, robust predictor of toxicity and T&O, no user objectivity as in cell counting and facilitates high throughput sample processing. Cons: Relatively expensive equipment and relatively technical to conduct

14 Comparison of microscopy and QPCR for estimating cyanotoxin risks Otten et al., Harmful Algae, In press.

15 Shotgun metagenomics for assessing microbial community structure and physiological potential

16 Assessment of microbial diversity and abundance

17 2013 transect caught a major geosmin event Cheney Reservoir: Wichita, Kansas Human perception of geosmin is 4 10 ng/l

18 Geosmin synthase

19 Development of a QPCR assay for geosmin detection Otten et al., In prep.

20 Application of the new QPCR assay to the 2013 geosmin event Otten et al., In prep.

21 Inference of MCs by microscopy, QPCR and metagenomics A) Time series of a Midwestern reservoir showing microcystin (MC LR eq.), Microcystis biovolume, toxigenic Microcystis by QPCR and shotgun metagenomics. B D) Linear regressions for each method relative to total microcystin. Otten et al., In prep.

22 Proposed method for CyanoHAB monitoring 1. Conduct bi weekly visual assessments of water. 2. Collect samples when algae becomes visible or bi weekly during peak months (e.g., May Oct) 3. Use QPCR to quantify potential toxin producers, if these > ~5,000 toxin genes/ml conduct appropriate ELISA Rationale: DNA based methods are cheaper ($50/sample/assay) and higher throughput than direct measurements of toxins ($75/sample/assay) and because most samples may contain low/no toxicity.

23 Thanks for your attention! Please feel free to contact me with any questions. Tim Otten, PhD, MPH Bend Genetics, LLC T: